1. Field of the Invention
This invention relates to aprotinin homologs and their production via recombinant DNA technology.
The chemically synthesized DNA molecules as disclosed herein are characterized by the DNA sequence coding for new polypeptides or polypeptides substantially agreeing in the amino acid sequence and composition of aprotinin or aprotinin homologs and having the biological activity of aprotinin or of aprotinin homologs.
2. Background Information
Aprotinin is a well known peptide comprising 58 amino acids and having the ability to inhibit trypsin, chymotrypsin, plasmin and kallikrein. Aprotinin is a basic proteinase inhibitor derived from bovine organs and has become a valuable drug, named Trasylol.RTM., for the treatment of various diseases like, e.g., hyperfibrinolytic hemmorrhage and traumatic-hemorrhagic shock (see H. Fritz and G. Wunderer, (1983), Drug. Res., 33, 479-494).
Recently it has been shown, that homologs of aprotinin with other aminoacids in position 15, instead of lysine, are valuable proteinase inhibitors having modified effects and efficacies in comparison to aprotinin (DE-OS 33 39 693; H. R. Wenzel et al, 1985, in Chemistry of Peptides and Proteins, Vol. 3). These aprotinin homologs have strong inhibitory effects on the elastases from pancreas and leukocytes, and on cathepsin G.
Such homologs of aprotinin can be used therapeutically in diseases in connection with excessive release of pancreatic elastase (pancreatitis), serum elastase (artherosclerosis), leukocyte elastase in acute and chronic inflammations with damage to connective tissue, in damage to vessel walls, in necrotic diseases and degeneration of lung tissue. Equally important is the part played by lysosomal enzymes, in particular leukocyte elastase, in inflammatory reactions due to immunological processes, for example, rheumatoid arthritis.
Although aprotinin and aprotinin homologs can be obtained from bovine organs and by semisynthetic conversion of the bovine trysin inhibitor (Tschesche, M., Wenzel, M., Schmuck, R., Schnabel, E., Offenlegungsschrift DE 33 39 693), the yields are relatively small.
It was perceived that the application of recombinant DNA and associated technologies would be the effective way of providing the necessary large quantities of high quality aprotinin homologs. The goal was to produce aprotinin homologs biologically active, as products of recombinant DNA technology from a host organism.
Methods for the expression of heterologous DNA in a microorganism are now known.
DNA coding for polypeptides of known amino acid sequences may be prepared by using the genomic DNA sequence, the cDNA sequence which is complementary to the mRNA or by choosing codons according to the genetic code and preparation of a synthetic gene.
A partial DNA sequence of a bovine genomic clone from bovine pancreatic trypsin inhibitor gene was cloned by S. Anderson and I. B. Kingston, (1983), Proc. Natl. Acad. Sci. USA, 80, 6838-6842 to characterize a genomic clone for BPTI.
A larger segment of the bovine genome coding for BPTI and bovine spleen inhibitor II were recently sequenced and published by Kingston, I. B. and Anderson, S., (1986), Biochem. J., 233, 443-450.